Antibiotic desideus and method of production



May 11, 1965 3,183,155

ANTIBIOTIC DESIDEUS AND METHOD OF PRODUCTION G. W. CAMIENER ETAL 3Sheets-Sheet 1' Filed April 10, 1962 od sd ed oed mod mad mod mwd mod Ndmuz E mama-mun manur- G. W. CAMIENER G. B. WHITFIELD P. F. WILEYINVHVTOR. a, 43%

ANTIBIOTIO DESIDEUS AND METHOD OF PRODUCTION Filed April 10, 1962 v May11, 1965 aw. CAMIENER ETAL 3 Sheets-Sheet 2 G. W. CAMIENER G. B.WHITFIELD P. F. WILEY INVENTOR.

BY 2 9b ATTORNEYS Mgy 1-1, 19.65 e. w. CAMIENER ETAL 3,183,

ANTIBIOTIC DESIDEUS AND METHOD OF PRODUCTION Filed April 10, 1962 sSheets-Sheet a CPS.,AT 6O MC.,FROM TMS.

G. W. CAMIENER G. B. WHITFIELD P. F. WILEY INVENTORS v A TTORNEYSSucrose United States Patent 3,183,155 ANTEBICTEC DESTDEUS AND METHOD CFPRCDUCTICN This invention relates to a novel composition of matter andto a process for the production thereof. More particularly thisinvention relates to a new compound, desideut, and to a process for theproduction thereof.

Desideus is a biosynthetic product obtained as an elaboration product ofa desideus-producing actinomycete. It is a neutral compound which has aproperty of adversely affecting the growth of certain organisms,particularly bacteria, for example, Bacillus subtz'lis, Bacillus cereus,and Streptococcus lactis, and tumor cells. Desideus can be used alone orin combination with other antibacterial or antitumor agents to preventthe growth of or reduce the number of such organisms or cells present invarious environments.

The actinomycete used according to this invention for the production ofdesideus has been designated as Streptomyces griseus var. desideus. Oneof its strain characteristics is the production of desideus. Asubculture of this variety can be obtained from the permanent collectionof the Northern Utilization and Research Division, Agricultural ResearchService, US. Department of Agriculture, Peoria, Illinois, USA. Itsaccession number in this repository is NRRL 2971.

Stre ton'zyces griseus var. desideus has yellowish gray aerial growthand yellowish gray or light to moderate olive reverse on most media.This organism has straight to flexuous spore chains as seen by the lightmicroscope and a smooth spore surface as seen by electron micrograph.Its growth characteristics on standard biological media and its carbonassimilation pattern are given in the following tables.

TABLE I.-A?PEARANCE ON EKTACHROME Agar Medium Surface Reverse 1.Bennetts Gray pink Yellowtan. 2. Cza-peks sucrese.. Whito--...Colorless. 3. Maltosetryptona. Gray pink Tan. 4. Peptone iron--Colorless Colorless. 5. 0.1 tyrosine. Trace gray pin i.-. Red tan. 6.Casein starch Gray pink Gray pink.

Dietz, A., Ektachrome transparencies as aids in actinomyceteelassification, Annals of the N.Y. Academy of Science 60: 152-154, 1954.

TABLE H.ASSIMILATION OF CARBON COM- POUNDS lN SYNTHETIC MEDIUMD-sorbitol *Pridham, T. G., and Gottlieb, D., Assimilation of CarbonCompounds in Synthetic Medium, J. Beet. 56 107414, 1948.

|- Positive assimilation.

- Negative assimilation.

() Slight growth-no assimilation.

(+) Positive asimilation-on1y slight growth.

ice

Patented May ll, 15%5 TABLE III.CULTURAL CHARACTERISTICS Medium Aerialgrowth Vegetative Other growth Plain gelatin. Trace white..- ColorlessComplete liquefaction.

Nutrient gelatin" .-...do o Do.

Nutrient nitrate Cream Yellow Surface ring.

broth. 2 cllow pigment.

No reduction. Synthetic nitrate ..---do Greenlsh Surface pelliele.broth. tinge Trace yellow pigment. No reduction.

Litmus milk Trace cream. Blue Surface ring.

Peptonization pH 7.8.

Peptone iron Pale pink..... Yellow tan... N 9 H28 darkenagar. mg.

Calcium malate Cream Cream M alate soluagar. bilized.

Skim milk agar..- Trace pink.... Y ellow tan Yellow tan pigment. Caseinhydrolyzed.

Glucose aspara- Cream pink-.. Cream None.

gine agar.

Casein starch Cream Cream pink-.. Starch hydroagar. lyzed.

Nutrient starch -..do Yellow Do.

agar.

Tyrosine agar. ..-..do Yellow tan..- Yellow tan pigment. Tyrosinesolubilized.

Xanthine agar..- Yellow Xanthine partially solubilized.

Maltosetryptone Olive brown- Yellow tan pigagar. merit.

Bennetts agan.-. Olive Czapeks sucrose Cream agar.

The new compound of the invention is produced when the elaboratingorganism is grown in an aqueous nutrient medium under submerged aerobicconditions. It is to be understood also that for the preparation oflimited amounts surface cultures in bottle can be employed. The organismis grown in a nutrient medium containing a carbon source, for example,an assimilable carbohydrate, and a nitrogen source, for example, anassimilable nitrogen compound or proteinaceous material. Preferredcarbon sources include glucose, brown sugar, sucrose, glycerol, starch,corn starch, lactose, dextrin, molasses, and like carbohydrate sources.Preferred nitrogen sources include corn steep liquor, yeast, autolyzedbrewers yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, panacreatic digest of casein, distillers solubles,animal peptone liquors, meat and bone scraps, and like nitrogenoussources. Combination of these carbon and nitrogen sources can be usedadvantageously. Trace metals, for example, zinc, magnesium, manganese,cobalt, iron, and the like need not be added to the fermentation mediasince tap water and unpurified ingredients are used as media components.

Production of the compound of the invention can be eifected at anytemperature conducive to satisfactory growth of the microorganism, forexample, between about 18 and 40 C. and preferably between about 26 and30 C. Ordinarily, optimum production of the compound is obtained inabout 2 to 10 days. The medium normally stays fairly close to neutral,or on the alkaline side, during the fermentation. The final pH isdependent, in part, on the buifers present, if any, and in part on theinitial pH of the culture medium which is advantageously adjusted toabout pH 6-8 prior to sterilization.

When growth is carried out in large vessels and tanks, it is preferableto use the vegetative form, rather than the spore form, of themicroorganism for inoculation to avoid a pronounced lag in theproduction of the new compound and the attendant inefiicient utilizationof the equipment. Accordingly, it is desirable to produce a vegetativeinoculum in a nutrient broth culture by inoculating the broth culturewith an aliquot from a soil or slant culture. When a young, active,vegetative inoculum has thus been secured, it is transferred asepticallyto large vessels or tanks. The medium in which the vegetative inoculumis produced can be the same as, or different from, that utilized for theproduction of the new compound as long as it is such that a good growthof the microorganism is obtained.

The novel compound of the invention is soluble in water-immiscible polarorganic solvents, for example, ethyl acetate, amyl acetate, butylacetate, and like aliphatic esters; l-butanol, Z-butanol, and likealiphatic alcohols; methyl ethyl ketone, methyl iso-butyl ketone, andlike alkanones; or chloroform, methylene chloride and like halogenatedhydrocarbons; and water-miscible organic solvents for example, methanol,ethanol, and like alcohols; and hydrocarbon solvents, for example,benzene and toluene; and is relatively insoluble in water.

A variety of procedures can be employed in the isola tion andpurification of desideus, for example, solvent extraction, liquid-liquiddistribution in a Craig apparatus, the use of adsorbents, andcrystallization from solvents. In a preferred process, desideus isrecovered from its culture medium by separation of the mycelium from thefiltrate, extraction of the mycelium with a water-miscible organicsolvent for desideus, and purification by tractional liquid-liquidextraction in a Craig counter-current distribution apparatus, using suchsolvent systems as Skellysolve B (isomeric hexanes):acetone:bulfer (pH7.5) 1752400275.

Further purifiication, to remove organic acids present, may be effectedby use of strongly basic anion exchange resins. Suitable anion exchangeresins for this purpose are obtained by chloromethylating by theprocedure given on pages 88 and 97 of Kunin, Ion Exchange Resins, 2nded. (1958), John Wiley and Sons, Inc., polystyrene crosslinked, ifdesired, with divinylbenzene prepared by the procedure given on page 84of Kunin, supra, and quaternizing'with trimethylamine, ordimethylethanolamine by the procedure given on page 97 of Kunin, supra.Anion exchange resins of this type are marketed under the trade namesDowex 2, Dowex 2-X8, Dowex 8, Amberlite IRS-400, Duolite A-102, andPermutit S1.

In accordance with a preferred procedure for the recovery of the newcompound of the invention the whole beer is adjusted, if necessary, to anear neutral pH or below and filtered. A filter aid, for example,diatomite, can be used. The clarified beer is discarded, and the washedmycelial cake is treated with a substantially water-miscible organicsolvent, for example, methanol to extract the desideus therefrom. Thevolatile solvent is removed from the extract in vacuo, and the aqueousresidue is extracted with a water-immiscible organic solvent, forexample, n-hexane, toluene, and ethyl acetate. The extract is passedthrough a chromatographic column, for example, a Florisil column (amixture of magnesium and sodium trisilicate), to adsorb the antibioticsubstance. The column can be developed with combinations of mixedsolvent systems, for example, n-hexanezacetone, acetone:- methanol, andmethanol. The fractions taken off the column are combined and evaporatedto dryness in vacuo to give a residue which is then stirred with awaterimmiscible solvent, for example, methylene chloride and filtered.The filtrates are then combined and filtered with the aid of a filteraid, for example, diatomaceous earth and then evaporated to dryness invacuo. The residue is dissolved in an equal volume of a water-immisciblesolvent, for example, n-hexane, and the solution refrigerated at a lowtemperature, for example, to C., until crystallization occurs. Aftercrystallization occurs, the crystals can be washed with a small amountof n-hexane at the low temperature and air-dried.

The new compound of the invention, desideus, has a broad antibacterialspectrum as shown in Table IV.

4 TABLE IV.ANTIBACTERIAL ACTIVITY OF The new compound of the inventionis active against Bacillus subtilis and can be used for treatingbreeding places of silk worms to prevent or minimize infections causedby this organism. It can also be used to minimize or prevent odor infish and fish crates caused by this organism. Further, the new compoundcan be used as a disinfectant on various dental and medical equipmentcontaminated with Staphylococcus aureus; it can also be used as adisinfectant on washed and stacked food utensils contaminated with thisorganism and the organism Streptococcus faecalis.

The following examples are illustrative of the process and products ofthe present invention, but are not to be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1 (A) Fermentation.-A soil stock of Streptomyces griseus var.clesz'deus, NRRL 2971, was used to inoculate a series of SOO-ml.Erlenmeyer flasks each containing ml of sterile seed medium consistingof the following ingredients:

Glucose monohydrate grarns 28 Milled cottonseed meal do 40 Tap water,q.s liters 1 The seed was grown for three days at 28? C. on a Gumprotary shaker operating at 250 r.p.m.

One shake flask of the seed described above (100 ml.) was used toinoculate a 40 liter seed tank containing 20 liters of sterile seed tankmedium consisting of the following ingredients:

Glucose monohydrate gram/liter 10 Pharmamedia do 2 Corns-teep liquor do10 Wilsons Peptone Liquor No. 159 2 do 10 Lard oil ml./liter 2 Tap waterBalance Glucose monohydrate gram/liter 20 Yeast do 2.5 Sodium chloridedo 3 Calcium carbonate do 4 Ammonium sulfate do 5 Kaysoy 1 do 10 Lardoil ml./liter 2 Tap water Balance Kuysoy is a fat-extracted,finelymil1ed, soybean meal.

The fermentor tank medium p-resterilization pH was 6.4. The fermentationcycle was four days during which time the temperature was controlled at28 C., filtered air was supplied at a rate of 100 standardliters/minute, and agitation was at the rate of 280 rpm. Sterile lardoil was added periodically to control foaming during the entirefermentation.

(B) Extraction.--The above described fermentation was run in duplicateand then the whole broth from the two fermentors was pooled, slurriedwith 4% diatomaceous earth and filtered. The filtered beer wasdiscarded. The mycelial cake was extracted four times with methanol andthe extracts concentrated in vacuo to an aqueous (20 liters). Theaqueous concentrate was then extracted twice with A volume ofSkellysolve B (trade name, Skelly Oil Company, Kansas City, Missouri,essentially nhexane B.P. 6068), four times with A volume of toluene andtwice with A volume of ethyl acetate. These organic extracts were eachconcentrated separately to an oil and the remaining aqueous concentratewas freezedried. These solvent extractions gave the followingpreparations:

Preparation Source Weight, g.

Skellysolve B extraction. 323. 4 Toluene extraction 179. 8 Ethyl acetateextraction. 78. 9

Spent aqueous dried... 855

Fractions Tubes Solids, g. Assn I'IBU/mg 1a (aqueous) 1, 130-1, 360 3.27 238 1b (solvent). 1, 130-1, 360 O. 55 172 2a (aqueous) 1, 360-1,460 1. 81 27 2b (solvent) 1, 360-1, 460 0. 17 152 *This tissue cultureassay is a mcausre of the inhibition of protein synthesis in a tissueculture using KB cells.

that dilution of 1 unit of material which elleets a 50% inhibition ofprotein synthesis 1000 KB U/unit of material (mg. or ml.)

One gram of fraction 1a from the above described CCD was dissolved inml. of methanol and passed through a column containing 30 g. of a 50100mesh, 8% divinylbenzene, eroslinked polystyrene, dimethyl ethanol benzylammonium anion exchange resin prepared according to US. Patent2,591,573. The column was prepared in the following manner. 30 g. ofanion exchange resin was slurried in methanol and poured into a 50 ml.burette. The substrate Was placed on top of the column and the columndeveloped with 75 ml. of methanol. The methanol was concentrated to aresidue giving 0.34 gram of a light brown viscous material. Thismaterial was then dissolved in 0.35 ml. of Skeilysolve B and chilled.The resultant crystals of the antibiotic desideus were washed with 0.5ml. of cold Skelly solve B and dried to give 0.16 gram (preparation PFN-109.1) having a melting point of approximately 47 C. and exhibiting anactivity of 4.75 B. subiz'lis units per milligram.

Additional crystals of desideus were prepared by using a Florisilcolumn. 450 grams of Florisil were slurried in Skellysolve B and packedinto a two inch TD. glass column. Fifteen grams of preparation one,previously described in Part B, were dissolved in methylene chloride andplaced on the column. The column wa eluted with one liquid holdup (ca.500 ml.) of each of the following solvent combinations:

Frlaption Solvent added Eluatc Wt., g

Slrcllysolve B (SSB) SSB SSE-acetone (8:2) SSB SSE-acetone (6iSSB-acetone (4:6)-

. SSE-acetone (4:)

SSE-acetone (2-8) Acetone SSE-acetone (2-8) Acctone-methanoM Acetone 8Acetone-methanol (6A).. Aegtgne-methanol 9 Acetone-methanol (4:6)Acetone-methanol 6: 10 Acet011e-Inethan0l(2:8) Acetone-methanol 11Methanol Acetogieanethanol 2:8 12 do Methanol 0. 58

Fractions 10, 11, and 12 were combined and extracted with three -ml.portions of methylene chloride. The combined methylene chloride extractswere filtered through celite, dried over anhydrous sodium sulfate andevaporated in vacuo to a residue (660 mg). This residue was dissolved in0.7 ml. of Skellysolve B and crystallization occurred. The crystals ofthe antibiotic desideus were collected and washed with cold SkellysolveB and dried to give 0.41 gram (preparation 113.15) having a meltingpoint range of 53-6l C. and exhibiting an activity of 5.0 B. subtilisunits per milligram.

The above Florisil procedure was repeated two more times to give acombined total of 0.84 gram for the three crystalline desideuspreparations.

The desideus crystals (0.16 gram) obtained via the Craig countercurrentand ion exchange resin procedure, as previously described, and thedesideus crystals (0.84 gram) obtained via the Florisil procedure,described above, were then combined and recrystallized by firstdissolving the crystals in Skellysolve B at a concentration of ca. 1gram/2 ml. The solution was filtered through a celite pad andrefrigerated at 15 C. The crystals were then removed by filtration andwashed with cold Siiellysolve B to yield 0.2 gram of desideus having amelting point range of 51-63 (3., no significant ultraviolet absorptionspectrum in the 220-320 mu range, a papergram with R values as shown inTable V- TABLE V Solvent systems: R values I 0.95

H 0.95 Ill 0.95 IV 0.95

I1-butanol, water (84: 16), 16 hours.

II1-butan01, water (84 16) plus 0.25% p-toluenesulfonic acid. 16 hours.

III1-butanol, acetic acid, water (2 1: 1), 16 hours.

IV2% piperidine (v./v.) in l-butanol, water (8 .1 16), 16 hours.

V1-butano1, water (4 96), 5 hours.

VI1butanol, water (4:90) plus 0.25% p toulenesulfonie acid, 5 hours.

and also as shown in FIGURE 1 of the accompanying drawing, an infraredabsorption spectrum expressed in reciprocal centimeters as follows andas shown in FIG- URE 2 of the accompanying drawing,

2920* Si (oil) 1060 S 2860 S 1020 M 1730 S (C O) 970 W 1690 W 943 W 1460S (oil) 936 W 1372 S (oil) 928 W 1328 M 908 W 1260 M 891 W Footnote onnext column.

1165 M 848 W 1150 M 811 W 1130 M 747 W 1115 S 718 W (oil) 1092 M*Frequency tolerances are :20 cm: in the 3800-2000 cm.- rang :10 cm. inthe 2000-1700 cm.- range, and 15 cm: in the 1700-700 cm. interval. Thespacing between adjacent bands shall be as indicated in the tabulationwith a tolerance of one-fifth of the frequency tolerance.

'tBand intensities are indicated as S, M, and W, respectively, andapproximated in terms of the backgrounds in the vicinity of the bands.An S band is of the same order of intensity as the strongest band in thespectrum M bands are between one-third and two-thirds as intense as thestrongest band, and W bands are less than one-third as intense as thestrongest hand. These estimates are made on the basis of a percenttransmission scale.

I and an elemental analysis as follows:

This tissue culture assay is a measure of the inhibition of proteinsynthesis in a tissue culture using KB cells.

that dilution of 1 unit of materia which effects a 50% inhibitionKBU/unit of material of protein synthesis (mg. or ml.) M 1000CRYSTALLOGRAPHY The X-ray diffraction pattern of crystalline desideusshows the following points in interplanar spacings, expressed inAngstrom units:

NUCLEAR MAGNETIC RESONANCE Desideus has a characteristic NMR spectrum asshown in FIGURE 3 of the accompanying drawing. The NMR spectrum wasobserved on a Varian A-60 spectrometer on a solution (ca. 0.5 ml., ca.0.3 molar) of the sample of desideus in deuterated chloroform. Thespectrum was calibrated against internal tetramethylsilane and theprecision of the Au was 11 c.p.s. Frequencies were recorded in c.p.s.downfield from tetramethylsilane.

(D) Desideus perchlrate.-Five hundred mg. of desideus, prepared as shownabove, was dissolved in ml. of methanol, and 0.5 ml. of 70 percentperchloric acid was added. The mixture was refrigerated overnight. Tenm1. of ether was added to the mixture to induce crystallization. Afterfiltration, 310 mg. of desideus perchlorate crystals having a meltingpoint of 153 C. were obtained. This product was recrystallized twicefrom methanol after which the melting point of the crystals increased to164 C.

ELEMENTAL ANALYSIS Calculated for C H O -HClO C, 57.37; H, 7.87; O,30.60; Cl, 4.23 (mol. wt.=837.4). Found: C, 56.52; H, 8.04; O, 30.78;Cl, 4.20 [mol. wt.:844 (calculated from C1 nt m- We claim:

1. A composition of matter assaying at least 25 mcg./

mg. of desideus, a compound which (a) is efiective in hibiting thegrowth of gram positive bacteria, and

(b) is effective in inhibiting the growth of KB cells in tissue culture;

and in its essentially pure crystalline form (0) is a neutral substance;

(d) is soluble in halogenated hydrocarbons, aliphatic alcohols,water-immiscible polar organic solvents, alkanones, and water-miscibleorganic solvents;

(c) has the following elemental analysis:

Calculated for O l-1 0 C, 65.21; H, 8.70; O,

26.09 Found: C, 65.42; H, 8.64; O, 25.85

(7) has a molecular weight as the perchlorate of 844;

g) has no significant ultravoilet absorption in the 220-320 m range; V p

(It) has a characteristic infrared absorption spectrum as shown inFIGURE 2 of the accompanying drawmg;

(i) has a melting point range of 5l-63 C.;

(j) has a characteristic papergram as shown in FIG- URE 1 of theaccompanying drawing;

(k) has a characteristic X ray diffraction pattern expressed in Angstromunits as follows:

and (I) has a characteristic NMR spectrum as shown in FIGURE 3 of theaccompanying drawing.

2. A novel compound, desideus, according to claim 1 in its essentiallypure crystalline form.

3. The perchlorate of desideus as defined in claim 1.

4. A process which comprises cultivating Streptomyces griseus var.desideus in an aqueous nutrient medium under aerobic conditions untilsubstantial desideus is produced.

5. A process which comprises cultivating Streptomyces griseus var.desideus in an aqueous nutrient medium containing a source ofassimilable carbohydrate and assimilable nitrogen under aerobicconditions until substantial desideus is produced and isolating thedesideus so produced.

6. A process according to claim 5 in which the isolation comprisesextraction of the desideus from the water wetmycelium with awater-miscible organic solvent.

A process which comprises (1) concentrating the extract obtained inclaim 6 to an aqueous solution, (2) extracting the aqueous solution witha water-immiscible organic solvent for desideus and (3) recoveringdesideus from the solvent extract.

8. A process according to claim 7 in which the recovery of the desideusfrom the solvent extract is accomplished by fractional liquid liquidextraction.

9. A compound as defined in claim 1, desideus, in its essentially pureform.

References Cited by the Examiner Antimicrobial Agents and Chemotheraphy,1961 (pages 169-177).

LEWIS GOTTS, Primary Examiner.

JULIAN S. LEVITT, Examiner.

UNITED STATES PATENT OFFICE CERTIFICATE 0F CORRECTION Patent No.3,183,155 1 May 11, 1965 Gerald W. Camiener et a1.

It is hereby certified that error appears in the above numbered patentrequiring correction and that the said Letters Patent should read ascorrected below.

Column 1 line 72, for "asimilat ion" read ---a-ssimilation column 2,line 46, for "panacreatic" read pancreatic column 6, line 42, for "0.2"read 0.32 column 7, line 29, for "materia" read material column 8, line4, for "hibiting" read inhibiting line 53, before "A process" insert 7.

Signed and sealed this 22nd day of November 1966.

( Amt:

ERNEST w. SWIDER Attesfing Officer EDWARD J. BRENNER Commissioner ofPatents

1. A COMPOSITION OF MATTER ASSAYING AT LEAST 25 MCG./ MG. OF DESIDEUS, ACOMPOUND WHICH (A) IS EFFECTIVE IN HIBITING THE GROWTH OF GRAM POSITIVEBACTERIA, AND (B) IS EFFECTIVE IN INHIBITING THE GROWTH OF KB CELLS INTISSUE CULTURE; AND IN ITS ESSENTIALLY PURE CRYSTALLINE FORM (C) IS ANEUTRAL SUBSTANCE; (D) IS SOLUBLE IN HALOGENATED HYDROCARBONS, ALIPHATICALCOHOLS, WATER-IMMISCIBLE POLAR ORGANIC SOLVENTS, ALKANNES, ANDWATER-MISCIBLE ORGANIC SOLVENTS; (E) HAS THE FOLLOWING ELEMENTALANALYSIS: CALCULATED FOR C40H64O12: C, 65.21; H, 8.70; O, 26.09 FOUND:C,65.42; H, 8.64; O, 25.85 (F) HAS A MOLECULAR WEIGHT AS THE PERCHLORATEOF 844; (G) HAS NO SIGNIFICANT ULTRAVOILET ABSORPTION IN THE 220-320 MURANGE; (H) HAS A CHARACTERISTIC INFRARED ABSORPTION SPECTRUM AS SHOWN INFIGURE 2 OF THE ACCOMPANYING DRAWING; (I) HAS A MELTING POINT RANGE OF51-63*C.; (J) HAS A CHARACTERISTIC PAPERGRAM AS SHOWN IN FIGURE 1 OF THEACCOMPANYING DRAWING; (K) HAS A CHARACTERISTIC X-RAY DIFFRACTION PATTERNEXPRESSED IN ANGSTROM UNITS AS FOLLOWS: 9.40 4.29 7.43 3.93 6.70 3.456.06 3.28 5.40 2.79 4.72